Inactivation is a form of molecular memory that is common to most potassium channels. The time course can range from slow (seconds) to ultra-fast, as can recovery; while multiple molecular mechanisms have been identified, those underlying inactivation of potassium channels in the heart remain incompletely understood. This Project will build on preliminary data we have developed to test hypotheses with respect to these mechanisms. We have found that secondary structure in the N-terminus chain of Kv1.4 is an crucial determinant of its inactivation by a ~ball-and-chain~ mechanism. In Specific Aim 1, we will use a structure-based deletion strategy to test the hypothesis that Kv4.3, recently- identified as a major component of human Ito, inactivates by similar mechanisms. In Specific Aim 2, experiments combining fluorescent imaging of modified channels with ion current measurements will be used to test the hypothesis that recovery from inactivation is initiated by movement of S4. We have identified a form of ultra-rapid inactivation in an N-terminus mutant of Kv1.4. In Specific Aim 3, we will test the hypothesis that this form of ultra-rapid inactivation also occurs in Kv4.3 and in the Ikr alpha-subunit, HERG. In addition, these mutant channels will be used to test the hypothesis that enhancement of pre-existing inactivation processes (rather than a ball-and-chain mechanism) underlies the effect of potassium channel beta- subunits. By elucidating the range of inactivation mechanisms present in cardia potassium channels, these experiments will provide further understanding of the determinants of the heterogeneous electrophysiologic behavior of the heart in health and disease.